PrimerExplorerV3_Manual_3

Advanced primer design

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381. Precautions in primer design 1.1 When too many primer sets are generated

a) Adjust the primer GC content.

When the primer GC content is 50 - 60%, favorable amplification performance will be obtained experimentally. Thus, the conditions are adjusted so that the GC content is in this range. Narrowing the permitted range for the GC content will be able to reduce the number of candidates. b) The differences in the Tm are set to about 5°C for the primers (regions F2 and F1c, regions B2

and B1c).

Under the principles of the LAMP reaction, the F2 of FIP (or B2 of BIP) anneals to the target gene and initiates the gene synthesis. The conditions are set so that F2 (B2) anneals to the target gene more frequently than F1c (B1c) does. For this reason, it is preferred that the Tm at region F2 (B2) be about 5°C lower than that at region F1c (B1c). When less stringent conditions (wider range of Tm’s at each primer location) are used to design the primers, primer sets are generated, which consists of the primers with various Tm value. For this reason, the difference in the Tm in each primer region may be 3°C or less. Also, best results are obtained if the Tm's match between regions F2 and B2, regions F1c and B1c, and regions F3 and B3. 1.2 When too few primer sets are generated

If only small number of primer sets is generated for GC rich or AT rich sequences, it is plausible that the primer design conditions for the given target sequence are too stringent. In PrimerExplorer V3, the primer design conditions are automatically selected for GC rich or AT rich sequences, but for some sequences, in spite of these conditions only a few primer sets are generated. In such cases, the range of primer length or the range of Tm should be adjusted. a) For AT rich sequences

For AT rich sequences, the Tm is calculated to be lower than non-AT rich sequences of the same length. For this reason the Tm based on the default primer length may be lower than the lower limit of default Tm value, and prevent primers from being designed. Thus, the primer length should be increased and/ or the Tm should be decreased. b) For GC rich sequences

In contrast, for GC rich sequences, the Tm is calculated to be higher than non-GC rich sequences of the same length. For this reason the Tm calculated from the default primer length may be higher than the default Tm upper limit, and prevent primers from being designed. Thus, the primer length should be decreased and/or the Tm should be increased. Because how the Tm or the length is adjusted would be determined on a case-by-case basis, the length of each primer should be changed by one base at a time and the Tm should be changed 1°C at a time. Once a large number of primers have been generated, then stop the adjustment and select the primers.

392. Designing wild type and mutant type primers In PrimerExplorer V3, it is possible to introduce mutations into the target sequence and then design primers. However, if there are too many mutations, the primer design conditions become too stringent and either the primers are not generated or the variety is insufficient. In such a case, one can design the primer with less stringent condition, for example reduce the number of the mutation point entered or completely eliminate the mutation sites from the target sequence. Appropriate primer sets could be selected, while identifying where the mutation points in the target sequence are located relative to the primer. 2.1 Detecting wild type and mutant type by amplification using common primers In general, the primers are design to exclude mutation within the primer region, but if there are numerous mutations, it may not be possible to design primers that satisfy these conditions. For this reason, primers are designed that allow (contain) mutations and if possible, try to design primers that are not likely influenced by the mutation. Under the principles of the LAMP reaction, F2 of FIP (or B2 of BIP) anneals to the target gene and initiates the gene synthesis. If the mutation is at the 3’ end of F2 (B2), the DNA polymerase has difficulty in recognizing the double strand formed between the primer and the target gene, thus inhibit the gene amplification. Similar principles apply to the 5’ end of F1c (B1c) and the 3’ end of F3 (B3). Therefore, primers are selected so that mutations are not located in these regions. On the other hand, if primers are selected so that the mutations are outside of the 3’ end in F2 (B2), 5’ end of F1c (B1c), or 3’ end of F3 (B3), the primers are less susceptible to the effect of the mutation and the both wild type and mutant type are detectable by a common set of primers. Thus, primers are selected by permitting the mutation to be located within the following locations (Table 2- 1). a) 3’ end of F1c or B1c and in the internal region b) 5’ end of F2 or B2 and in the internal region c) 5’ end of F3 or B3 and in the internal region Here, we will design common primers that detect M13 and its mutant. Figure 2-1 shows an alignment of the wild type and the mutant type. In the entire length of 510 bp there are seven mutations. The region containing these mutations is the target region for amplification. Figure 2- 2 shows an example of primer selection. Under default, primers have been designed for the wild type strain. Here, we focus on the 25 primer set candidates that contain the mutations and select the common primers. A star shows the locations of the mutations, and the locations of the mutations applicable to the primers designed are enclosed by a dotted line. This can be used to confirm the location of the primers for a given mutation. Table 2- 2 shows these results. The black dot indicates the location of the primer region (5’ end, internal region, 3’ end) for each primer set (F3, F2, F1, B1, B2, B3) for a given mutation. It can be determined that No 1 - 5, No 9 - 13, and No 25 are the primers less susceptible to the effects of the mutations at amplification. These are selected from the primer list, and their detailed information is then assessed for a final selection of the primer sets.